With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. In particular, next generation therapeutics derived from monoclonal antibodies (e.g. Fab, scAb, scFv, immunotoxins, etc) show great clinical promise in treating a range of human disorders including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Currently, the bacterial Sec protein export pathway is the dominant mechanism for protein display (e.g., phage display, cell surface display) and also for the expression and engineering of small, non-glycosylated antibody fragments. However, due to a number of limitations associated with Sec export, bacterial production of stable, high-affinity antibody fragments in high yields for preclinical and clinical trials can be a serious bottleneck in the antibody drug pipeline. Therefore, the overall objective of the proposal is to develop a versatile antibody expression and engineering platform by capitalizing on the remarkable properties of the recently discovered bacterial twin-arginine translocation (Tat) pathway. Towards this objective, the current proposal encompasses the following specific aims: (1) creation of synthetic libraries of single-chain Fv (scFv) antibody fragments;and (2) application of a proprietary Tat-based genetic selection strategy for efficient isolation of super-stable, ultra- high affinity human scFvs against disease-relevant antigens. The proposed studies are expected to result in a rapid, single-step selection platform for the discovery of scFv sequences that fold and function in intra- or extra-cellular environments. Successful completion of the proposed studies is expected to result in a powerful new technology for generating potent antibody-based biopharmaceuticals, diagnostics and research reagents. PUBLIC HEALTH RELEVANCE: By 2008, engineered antibody fragments are predicted to account for >30% of all revenues in the biotechnology market and will be used to treat a wide array of human diseases including bacterial and viral infections, cancer, inflammatory diseases and neurodegenerative disorders. Since antibody therapies are an increasingly large fraction of the drugs in development, with ever escalating increases in the cost of drug development, any improvements to the production or discovery of efficacious antibody fragments will have a significant impact on human health. Thus, this proposal seeks to develop a novel bacterial selection strategy for rapid, low-cost isolation of potent human antibodies against virtually any target antigen of interest.